Biofuel milking Tuan Nguyen & Rupak Doshi Geoffrey Chang Lab - - PowerPoint PPT Presentation

Objectives: Use of structure-based/protein engineering methods to develop a system capable of secreting oil. In principle, this system could “plug-and-play” with a variety of oil-producing

• rganisms for secreting biofuel.

AFOSR 2012 Bioenergy Program Review August 6-10, 2012 – Arlington, VA

Tuan Nguyen & Rupak Doshi

Geoffrey Chang Lab Department of Molecular Biology The Scripps Research Institute

Transpor ansporter ter-media mediated ted Biofuel “milking”

Outline

• 1. Biofuel milking: why and how?
• 2. Membrane proteins and ABC-transporters
• 3. Experimental strategy
• 4. Selection of transporter candidates
• 5. Selection of substrate candidates
• 6. Construction of E. coli strains for transport assay
• 7. In vitro transport assay
• 8. In vivo transport assay (intact cell & spheroplast)
• 9. Future directions

Tuan Nguyen Rupak Doshi

• Certain ABC transporters can export biofuel substrates
• Secretion could lead to higher yields of biofuels

Current Biofuel Platforms for Commercial Production

“Farming” (sunlight harvesting using algae) Sapphire Energy, PetroAlgae, Synthetic Genomics… “Fermentation” (mixotrophic algae, yeast…) Solarzyme, Amyris Biotechnologies, LS9, Coskata…

Harvesting & extraction of biofuels is energy-intensive! From our pharmacology perspective: How do cells transport hydrophobic oil-like molecules across the lipid bilayer? Develop a “plug-and-play” secretion system that is energy-efficient and sustainable.

Kruwlich et al., 2005. 3, 566-572

MsbA (ABC)

Andrew Ward Jodie Yu Christopher Reyes Christopher Roth

EmrD (MFS)

Yong Yin Xiao He That Nguyen Paul Szewczyk

NorM (MATE)

Xiao He Paul Szewczyk Andrey Karyakin Miriah Hevin Wen-Xu Hong Qinghai Zhang

EmrE (SMR)

Yen-Ju Chen Owen Pornillos Alex Ma Samantha Lieu Andy Chen Andrew Ward Rupali Aggarwal Alexandra Caya Steve Aller Jodie Yu Qinghai Zhang (TSRI) Yue Weng Srinivas Chittaboina Ina Urbatsch (TTUHSC) Rupeng Zhuo Patrina Harrel Yenphuong Trinh

P-glycoprotein (ABC)

The ABC-transporter family

• structure and function -

Structure Hydrophobic substrate binding model

Diversity of molecules recognized by Pgp (ABCB)

The ABC-transporter family

• diversity of substrates -

The ABC-transporter family

• classification -

Family Characteristics Function

ABCA

Very large in size Transport cholesterol and lipids.

ABCB

Contain both full & half transporters Transport lipids and diverse drug substrates

ABCC

Full transporters Ion transport, toxin secretion

ABCD

Half transporters Only in peroxisomes

ABCE/F

Contains only ABC domain and no transmembrane domain Not actual transpoters

ABCG

Half transporters in reverse

• rientation

Transport lipids, drugs, bile, cholesterol and other steroids

Transporter-mediated biofuel “milking”

“Substrates”

Terpenoids (isoprenoids): MVP & MEP pathways

• Monoterpenes (C10, limonene, camphor, carvone,

eucalyptol)

• Triterpenes (C30+, squalene, botryococcenes)
• Tetraterpenes (C40, carotene, lycopenes)

Wax, paraffin: very long chain alkanes (CnH2n+2) Triacylglyceride (TAG, fat):

“Pumps” (ABC transporters)

ABCB family:

• 1. Lipid flippase MsbA (9 bacterial orthologs)
• 2. Lipid flippase MDR3/ABCB3)
• 3. “Drug” transporters (Pgp/MDR1/ABCB1)
• 4. B. braunii transcriptome ABCB ORFs
• ORF #7809
• ORF #33468
• ORF #26423
• ORF #24910

ABCG family:

• 1. Cholesterol excretion (ABCG5/8)
• 2. Plant wax transporters (wbc11/12)
• 3. B. braunii transcriptome ABCG ORFs
• ORF #24393
• ORF #19521
• ORF #7846
• ORF #33471

Screen and re-engineer potential transporters for binding and transport, focusing mostly on ABCB and ABCG family Identify biofuel molecules where the metabolic pathway and hosts are well-established.

Screening of transporter candidates

Plant Wax Transporter

• CER5 and WBC11 (WBC11/12) form

a heterodimer to transport long chain hydrocarbons (Samuels et al., Annu. Rev. Plant

Biol, 2008).

• EIBI1 full-size ABCG wax transporter

(Chen et al, PNAS 2011).

Transporter Descriptions Progress

• Genes codon-optimized & synthesized
• Expressed & screened in Pichia
• Found no clone with good expression

for WBC11/12

• Eibi1 screening is underway
• B. braunii transcriptome ABCBs

and ABCGs ABCB: 7809, 33468, 26423, 24910 ABCG: 24393, 19521, 7846, 33471

• ORFs of ABCB & ABCG homologs

identified from B. braunii transcriptome

• Genes codon-optimized & synthesized
• Expressed & screened in Pichia
• Large-scale expression and purification

testing is underway

Bacterial & mamalian ABCBs MsbA and its homologs PGP

• Obtain 19 MsbA homolog constructs in

pET19b

• ATPase assay against a variety of

terpenoids

• In vitro and in vivo transport assay

Rupak’s talk

Screening of substrate candidates

Liquid fuels (Botryococcenes…) Gene SSL1/3 responsible for botryococcenes synthesis identified (Niehaus et al.,PNAS, 2011).

Substrate Descriptions Progress

• SSL1 and SSL3 cloned in E. coli

pET19b (IPTG induction) and pBAD28 (arabinose induction).

• In vitro synthesis of Botryococcenes

using FPP and SSL1/3 is underway.

Carotenoids Lycopene, carotene, canthaxanthin, phytoene, zeaxanthin, etc…

• Obtain 8 constructs for carotenoids

expression in E. coli (Dr. Cunningham,

• Univ. of Maryland).
• Create BL21 E. coli strains expressing

both carotenoids and MsbA homologs

• In vitro and in vivo transport assay

Rupak’s talk

MVA, MEP, botryococcenes & carotenoid pathways

GPP (C10) FPP (C15) Squalene (C30) PSPP (C30) Botryococcenes (C30)

FPP synthase SSL1 SSL3 SSL2 BSS

SSL1 + SSL2 = squalene SSL1 + SSL3 = botryococcenes (Niehaus et al.,PNAS, 2011).

Carotenoid-expressing E. coli strains

ipi crtE crtB crtI crtZ crtY ipi crtE crtB crtI bkt crtY ipi crtE crtB crtI crtY ipi crtE crtB crtI lcyE2 crtE crtB crtI lcyE1 ipi crtE crtB crtI ipi crtE crtB ipi crtE

Constructs Phenotype colorless colorless red yellow yellow yellow pink yellow

• 1. GGPP ipi
• 2. Phytoene ipi
• 3. Lycopene ipi
• 4. Delta-carotene
• 5. Epsilon-carotene ipi
• 6. Beta carotene ipi
• 7. Canthaxanthin ipi
• 8. Zeaxanthin ipi

(Adapted from Cunningham & Gantt, 2007)

• E. coli strains for transport assays

Pair-wise double transformation to E. coli BL21 strain Transport assays IPTG induction

Features Carotenoid-expressing constructs Transporter-expressing constructs

Backbone pAC184 pET19B Antibiotic resistance CmR AmpR Origin of replication p15A pBR322 Promoter/Induction Constitutive / None T7 / IPTG